ANTI-PLASMODIAL QUINONES FROM THE RHIZOMES OF KNIPHOFIA FOLIOSA

Transcription

1 ANTI-PLASMDIAL QUINNES FRM THE RHIZMES F KNIPFIA FLISA MARTHA KHASIALA INDULI BSc. (Moi Univ), MSc. (UoN) SUPERVISRS: Prof. Byamukama Robert Makerere University Prof. Abiy Yenesew University of Nairobi 8 TH CTBER,

2 Malaria An endemic and deadly disease(casteel, 2003) Been noted for more than 4000 years Vector: The female Anopheles mosquito (Pillay et al., 2008) Causal agent: Protozoal micro-organisms of the genus Plasmodium (Pillay et al.,2008) 2

3 Malaria: statistics Distribution: distributed in 101 countries, 45 in sub-saharan Africa(Casteel, 2003) Between million cases of malaria infections every year(w, 2009) million deaths every year (Casteel, 2003) In Africa south of Sahara,75% of deaths are children under five (Africa Malaria Report, 2003) 3

4 Malaria: More information Major public health and economic problem in tropical and sub-tropical region(kaur et al. 2009) Besides causing up to 2 million deaths, causes unimaginable suffering and disability and leads to economic loss (Sachs and Malanay, 2002) 4

5 Prevalence of malaria Taylor et al

6 PRBLEM STATEMENT 6

7 Anti-malarial drugs Resistance Plasmodium strain Resistance first reported (year) Reference Quinine P. falciparum 1910 ICMR bulletin, 2008; Gregsonand Plowe, 2005; Bloland, 2001; Chloroquine P. falciparum 1960 ICMR bulletin, 2008; Bloland, 2001 P. vivax 1989 ICMR bulletin, 2008; Mendis et al., 2001 Sulphadoxine /pyrimethamine P. falciparum 1967 ICMR bulletin, 2008; Bloland, 2001 Mefloquine P. falciparum 1981 ICMR bulletin, 2008; Bloland, 2001 Artemisinin P. falciparum - World malaria report, 2009 Resistance due to the indiscriminate/inappropriate use of drugs, non compliance and the close structural relationship of anti-malarial drugs in use

8 Main objective of the study To search for anti-malarial bioactivities of the crude extracts and isolated secondary metabolites from the rhizomes of Kniphofia foliosa 8

9 Specific objectives of the study 1. To collect, extract and isolate secondary metabolites from the rhizomes of Kniphofia foliosa 2. To carry out structural elucidation of the isolated secondary metabolites using various spectroscopic methods 3. To establish the in vitro anti-plasmodial properties of the crude extracts, and isolated secondary metabolites and compare them with the current anti-malarial drugs 4. Establish the in vivo anti-plasmodial properties of the isolated secondary metabolite with good in vitro anti-plasmodial activity 9

10 JUSTIFICATIN 10

11 Why the focus on drug development as a solution to the malaria problem? Due to the persistent problem of resistance, there is urgent needfor new anti-malarial drugsthat are both effectiveand affordable. Also in order to delayonset of resistancethere is need for a poolof effectiveanti-malarial drugs with different modes of action. To avail cheap and effective herbal drugs 11

12 Why the focus on plants as a solution to the existing malaria problem? H Me H H N H H N Quinine From Cinchona succuriba Artemisinin From Artemisia annua Use of natural products with therapeutic properties as ancient as human civilisation with plants being a main source 12 of drugs (Rates, 2001)

13 Why the focus on plants belonging to the genus Kniphofia K. foliosa (Wube et al., 2006) Some species belonging to the genus Kniphofia are used in ethno-medicine for the treatment of malaria 13

14 Why the focus on metabolites belonging to the genus Kniphofia H H CH 3 H H H CH 3 H Chrylandicin 0.54 (3D7 strain) Wube et al., 2005 H CH 3 CH 3 Knipholone 1.06 (K1 strain) Bringmann et al.,2008a Secondary metabolites with promising anti-plasmodial activity isolated from Kniphofia foliosa plant CH 3 14

15 Scope of study The plant considered for this study was Kniphofia foliosa The selection of this plant was based on the ethno-medical uses and chemotaxonomic/ bioassay information from the available literature(wube et al., 2006; Bringmann et al., 2008a). 15

16 Botanical information on Asphodelaceae Asphodelaceae Two sub-families 17 genera, 750 species Asphodeloideae 11 genera, app. 261 species Alooideae 7 genera Kniphofia Bulbine Aloe app. 400 species, 83 in East Africa. 60 in Kenya 70 species in Africa, 1 in Kenya (K. thomsonii) K. foliosa perennial herb endemic to Ethiopia 41 species in Southern Africa, 2 species in Kenya 16 (Smith and Van Wyk, 1991)

17 Ethno-medical information on some species of the genus Kniphofia Species Plant part Used for/ As Reference K. foliosa Roots Abdominal cramps and aches Wound healing K. linearifolia Roots To treat infertility in women K. isoetifolia Roots Gonorrhea, hepatitis B K. laxiflora Plant infusion Snake deterrents; chest aliments Wube et al., 2005 Wube et al., 2005 Bosch, 2008 Yineger et al., 2008 Ramdhani, 2006; Bringmann et al.,

18 Some biological activities of metabolites of the genus Kniphofia Biological activity Compound Reference Antioxidant activity Knipholoneanthrone Habtemariam, 2007; Bringmann et al., 2008a Antiprotozoalactivity, Knipholone anthrone Habtemariam, 2007 radical scavenging effect against DPPH Antitumour activities Knipholone (HSC-2 cells) isoknipholone Bringmann et al., 2008a Antimalarial (K1 and Isoknipholone NF54 strains of Knipholone anthrone Plasmodium falciparum) 18

19 Phytochemistry of Kniphofia H H CH 3 H H H CH 3 H CH 3 Islandicin Berhanu et al., 1986 H H Chryslandicin Dagne et al., 1987 H H 1 8a 1a H Me H a 4a Me H 1' H 6' 3' Me Me Me Me Knipholone Yenesew et al., 1994 Knipholone cyclooxanthrone Abdissa et al.,

20 METDLGY 20

21 verview of Methodology Field collection: Rhizomes of K. foliosa plant collected from the Chemistry Department, Addis Ababa University garden, Ethiopia in November, 2009 Extraction: (solvents used DCM:MeH (1:1) and MeH) Isolation and Purification: (Column chromatography, Sephadex(LH-20), Prep. Thin layer chromatography) Structure elucidation: ( 1 H and 13 C NMR, UV, Mass Spectroscopy) Bioassays: in vitro and in vivo anti-plasmodial assays (Yenesew et al., 2012)- in collaboration with KEMRI 21

22 Plant collection and processing 22

23 Extraction and isolation of compounds Led to the isolation of 14 compounds 23

24 Characterization of compounds At the University of Nairobi 1 H (200 MHz) 1 3 C NMR (50 MHz) Additional data to be generated: UV-VIS SPECTRA IR SPECTRA MELTING PINT Me Me 24

25 University of Mississippi s Natural Products Laboratory HIGH FIELD NMR CD SPECTRMENTER HRMS University of Gothenburg, Sweden University of Botswana 25

26 Biological tests Anti-plasmodial activity i(n vitro) Antimalarial activity (in vivo) 26

27 RESULTS AND DISCUSSIN 27

28 Compound 1 Physical properties: obtained as a pale yellow amorphous solid that intensifies in colour upon exposure to ammonium vapour Eluted with 15% EtAc in n-hexane Has an Rf value of 0.3 in 100% dichloromethane 28

29 UV data of Compound 1 UV data: (λ max 284, 360 and 412 nm) which is indicative of a phenylanthrone moeity

30 1 H NMR (Expansion) Spectrum Data for Compound 1 H H C B A 7.62, t, J=7.8 Hz H , dd, J=1.2,7.8 Hz H , s, H , dd, J=1.2,7.8 Hz H-5 H 8 9 H 1 C B A CH 3 δ C δ H 2.38 Suggestive of the presence of a chrysophanol anthrone moiety

31 1 H NMR (Expansion) Spectrum Data for Compound , s, H ,s CH 3-4 CH 3-3 H H C B A CH 3 2' H 4' CH 3 Suggests the presence of an acetylphloroglucinol methyl ether substituent

32 13 C NMR Spectrum Data for Compound 1 H H C B A 76.7, -sp 3 C 5 10 H 4 2' CH 3 H 4' CH 3 13 C NMR spectrum showed the presence of twenty seven non-equivalent carbon atoms

33 1 H NMR (Expansion) Spectrum Data for Compound 1 cont. 4.00,s CH 3-4 CH 3-3 CH , d, J=15 Hz 2.87, d, J=15 Hz CH 2-1 CH 3-3

34 HSQC Spectrum Data for Compound 1 δ H 3.17, d, δ H 2.87, d δ C 51.1

35 13 C NMR Spectrum Data for Compound 1 H H δ C δ C C-3 δ C C-9 C 5 B 10 H A 4 2' CH 3 H 4' CH 3

36 HMBC Spectrum Data for Compound 1 δ H 1.64 δ H 3.17, d δ H 2.87, d δ C Correlation between the methylene and methyl protons with the carbonyl at (δ C 203.7) confirms the presence of an acetonyl substituent

37 HMBC Spectrum Data for Compound 1 δ H 3.17, d δ H 2.87, d H H C 5 B 10 H A 4 2' CH 3 H δ C ' CH 3 Correlation between the methylene protons of the acetonyl substituentandc-10(δ C 76.7)confirmstheplacementofthe acetonyl substituent at C-10

38 HRMS data of Compound 1 SA-6U, MW=492 Heydenreich_81 #6-227 RT: AV: 222 NL: 8.08E5 T: + c Full ms [ ] H 8 9 H 1 Relative Abundance C 5 B 10 H A 4 4' 2' CH 3 H CH m/z The molecular ion peak at m/z correspondingtomolecularformulac 27 H 22 8

39 1 H NMR Spectrum Data for Compound 1 H H Three Chelated H groups C 5 B 10 H A 4 2' CH 3 H 4' CH 3 The lack of H signals (except for the chelated hydroxyl groups) even in acid fee solvent is evident

40 Final structure H H C B A CH 3 2' H 4' CH 3 By further making reference to Abdissa et al., 2013, compound 1 characterised as 10- acetonylknipholone cyclooxanthrone.

41 ther compounds isolated from this study Anthraquinones H H H H H H Ac Chrysophanol Aloe-emodin acetate Berhanu et al., 1986 Berhanu and Dagne 1984 Deoxyerythrolaccin Yagi et al., 1974 H H H CH 3 H CH 3 H H Islandicin Berhanu et al., 1986 Laccaic acid D DNP,

42 ther compounds isolated in this study cont. Anthraquinone dimers CH 3 CH 3 H Me H H H H CH 3 H Chryslandicin 10 methyl ether H CH 3 H 3 C H Chryslandicin Dagne et al., 1987 H H H 3 C H Asphodelin Adinolfi et al.,

43 ther compounds isolated in this study cont. Phenylnthraquinones/ anthrones H H H H CH 3 Me H H CH 3 CH 3 Me Me Knipholone Yenesew et al., 1994 Knipholone anthrone Yenesew et al.,

44 ther compounds isolated in this study cont. Phenylnthraquinone dimers S H Me P H Me Me S H Me P H Me Me P Me Me Me M Me Me Me Joziknipholone A Bringmann et al., 2008b Joziknipholone B Bringmann et al., 2008b 44

45 ther compounds isolated in this study cont. Minor phenolic (benzoic acid derivative) H H 3,4-dihydroxybenzoic acid 45

46 Chemotaxonomic significance of this study This is only the second report on the occurence of the dimeric phenylanthraquinones Joziknipholones A and B in nature having previously been isolated from the roots of Bulbine frutescens (Bringmann et al., 2008b) Deoxyerythrolaccin, laccaic acid D, asphodelin and 3,4-dihydroxybenzoic acid are reported for the first time from the genus 46

47 in-vitro antiplasmodial activities of crude extracts and selected compounds of the rhizomes of K. foliosa against (D6) and (W2) strains of P. falciparum Sample Crude extracts IC 50 values (µg/ml ± SD) K. foliosa (DCM:MeH extract) 3.4± ±0.2 K. foliosa(meh extract) 4.7± ±0.8 Dimeric anthraquinones Chryslandicin 2.1± ± hydroxy-10-(chrysophanol-7 -yl) chrysophanol anthrone D6 W2 1.7± ±0.2 Asphodelin 8.2± ±1.4 KEY: D6: Chloroquine sensitive W2: Chloroquine resistant (Yenesew et al., 2012) 47

48 in-vitro antiplasmodial activities of crude extracts and selected compounds of the rhizomes of K. foliosa against (D6) and (W2) strains of P. falciparum cont. Sample Phenylanthraquinones/ anthrones IC 50 values (µg/ml ± SD) Knipholone 10.1± ±0.5 Isoknipholone 8.6± ±1.2 Knipholone anthrones 4.1± ± Acetonylknipholone cyclooxanthrone 4.4± ±1.2 Dimeric phenylanthraquinones Joziknipholone A 0.4± ±0.1 Joziknipholone B 2.5± ±0.2 Standard drugs Chloroquine 0.007± ±0.03 mefloquine 0.03± ± D6 W2 48

49 in vivo antiplasmodial activity for knipholone anthrone isolated from the rhizomes of Kniphofia foliosa Sample Dose mg/kg/day Parasitemia Mean (SD) value % Knipholone anthrone (2.3) Quinine hydrochloride Chemosupression in an in vivo 4-day Plasmodium berghei ANKA suppressive test at 100 mg/kg/day; knipholone anthrone showed marginal activity with 30.13% chemosupression being observed 49

50 Conclusion A total of fourteen (14) compounds have been isolated from the rhizomes of K. foliosa of which two namely 10 acetonylknipholone cycloanthrone (1) and chryslandicin- 10-metyl ether are novel The dimeric phenylanthraquinone Joziknipholone A showed the highest activity with IC 50 values of 0.4±0.1 and 0.3±0.1 µg/ml against D6 and W2 strains 10 acetonylknipholone cycloanthrone (1) exhibited significant activity with an IC 50 value of 3.1±1.2 µg/ml against the chloroquine resistant (W2) strain Marginal activity observed for knipholone anthrone (30.13% chemosupression) in an in vivo 4-day P. berghei ANKA suppressive test at 100 mg/kg/day 50

51 Recommendations/ Further work To test the pure enantiomeric forms or at least the enantiomerically enriched forms of the phenyantraquinones in order to establish the relationship between configuration and antiplasmodial activity Further phytochemical work on other species of Kniphofia To test the isolated compounds against a wider range of micro-organisms (both anti-bacterial and anti-fungal) and other strains of P. falciparum 51

52 Publications to date Induli M., Gebru M., Abdissa N., Akala H., Wekesa I., Byamukama R., Heydenreich M., Murunga S., Dagne E., Yenesew A. (2013) Antiplasmodial quinones from the rhizomes of Kniphofia foliosa, Natural Products Communication, 8, p Induli, M., Cheloti M., Wasuna A., Akala H., Wekesa I, Wanjohi,J.M., Byamukama R., Heydenreich M., Makayoto M., Yenesew A. (2012) Naphthaquinones from the roots of Aloe secundiflora, Phytochemistry Letters, 5, p

53 Acknowledgements DAAD Deutscher Akademischer Austausch Dienst German Academic Exchange Service 53

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